Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed for Angiopoietin-2 (Angpt-2) from endothelial cell culture supernatants and endothelial cell lysates with the commercial human Angiopoietin-2 DuoSet kit (DY623, R&D Systems, Minneapolis, MN). All additionally needed reagents were purchased from R&D Systems except for Normal Mouse Serum (NMS) (Jackson ImmunoResearch Laboratories, Westgrove, PA) and Bovine Serum Albumine (BSA) (Sigma-Aldrich, St. Louis, MO).
Cell culture studies
Primary endothelial cell isolation was performed according to institutional and governmental guidelines. In brief, human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical veins. HUVECs were isolated with heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA), Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA) and Collagenase (Biochrom, Berlin, Germany). Then, they were grown to confluency in endothelial growth medium (EBM-2) containing 2% FBS according to manufacturer’s instructions (Lonza, Basel, Switzerland). For HUVEC donation, informed consent was obtained and the protocol was approved by the local ethical committee of Hannover medical school (No. 1303–2012).
HUVECs were used in passages 3–5 and split with Trypsin/Ethylenediaminetetraacetic acid solution (Biochrom, Berlin, Germany), FBS and PBS.
Unless otherwise specified, HUVECs were stimulated with 10 µM Bifonazole (Sigma-Aldrich, St. Louis, MO), 10 ng/ml recombinant human TNF-alpha (R&D Systems, Minneapolis, MN), 50 ng/ml Phorbol-12-myristate-13-acetate (PMA) (Merck Millipore, Darmstadt, Germany), 10 µM DMSO for molecular biology (Sigma-Aldrich, St. Louis, MO), 10 µM Mibefradil dihydrochloride hydrate (Sigma-Aldrich, St. Louis, MO), 50 µM TTA-A2 (Sigma-Aldrich, St. Louis, MO), 1 mM Nω-Nitro-L-arginine methyl ester hydrochloride (L-Name) (Sigma-Aldrich, St. Louis, MO) and 1 U/ml Thrombin (Merck Millipore, Darmstadt, Germany). Each condition was reproduced for n = 4–10 times.
Antibodies and reagents
All reagents were purchased from Sigma Aldrich (St. Louis, MO) unless otherwise specified. Antibodies against Angpt-2 (AF623) (R&D Systems, Minneapolis, MN), vWF (A008202) (Agilent Dako, Santa Clara, CA), VE-Cadherin (555661) (BD Pharmingen, San Jose, CA), Phalloidin/F-Actin (A22283) (Invitrogen, Carlsbad, CA) were utilized. As secondary antibodies goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, CA) and donkey anti-goat IgG-HRP (Santa Cruz Biotechnology, CA) were used.
Fluorescent immunocytochemistry
Coverslips were coated with collagen (Sigma-Aldrich, St. Louis, MO) and HUVECs were grown to confluency. Then coverslips were fixed with a 1:1 solution of Aceton (Thermo Fisher Scientific, Waltham, MA) and Methanol (Th. Geyer Hamburg, Hamburg) or 2% Paraformaldehyde (Th. Geyer Hamburg, Hamburg). All coverslips were then blocked with 10% donkey serum (Jackson Immuno Research Inc., West Grove, PA) and those fixed with 2% Paraformaldehyde were additionally permeabilized with 0.1% Triton X-100 in PBS (Sigma-Aldrich, St. Louis, MO). The primary antibody was incubated for 1 h at room temperature, followed by washing with Phosphate buffered saline (PBS) (Thermo Fisher Scientific, Waltham, MA). The secondary antibody was incubated for 1 h at room temperature. Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA), Alexa Fluor 555 donkey anti-goat IgG (Thermo Fisher Scientific, Waltham, MA) and Alexa Fluor 556 donkey anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA) were used as secondary antibodies.
Pictures were taken with a Leica DMI 6000B microscope and obtained with the same light exposure conditions and gain.
RNA –Isolation and quantitative PCR
RNA was isolated from cultured HUVECs using the RNeasy MicroKit (Qiagen, Hilden, Germany) following manufacturer’s instructions. With the transcriptor First Strand cDNA Synthesis (Roche Diagnostics, Rotkreuz, Switzerland), 0.5 µg of total extracted RNA was reverse transcribed to cDNA. After that, SYBR Green real-time-PCR using a LightCycler 480II (Roche, Basel, Switzerland) was performed. The following primers were used: human ß-Actin (fv: CTG GAA CGG TGA AGG TGA CA, rev: AGT CCT CGG CCA CAT TGT G), human Ang-2 (fv: GCC GCT CGA ATA CGA TGA CT, rev: GCT TCA TTA GCC ACT GAG TGT TGT). For each sample triplicates of RT-qPCR were performed and average of the cycle values was formed.
Transendothelial electrical resistance (TER)
Special cell culture plates (ibidi, 8W10E) were coated with collagen for 1 h at 37 °C and HUVECs were grown at 37 °C. Monolayer confluency was determined regarding manufacturer’s recommendation by electrical criteria (resistance > 1800 ohms and capacitance > 10 nF). HUVECs were stimulated with 10 µM BIFO for 24 h. Then 1 U/ml Thrombin was applied. Medium was changed by carefully removing the medium and renewing it by not disturbing the cell monolayer in these steps. Transendothelial electrical resistance (TER) was measured at different time points by an electric cell-substrate impedance sensing system (ECIS, Applied BioPhysics Inc.). Values were either plotted over time or as bar graphs at time points of maximal response as described elsewhere [37, 38]. Resistances of each condition at each time point were divided by its condition starting resistance to calculate normalized TER.
Transwell Assay
HUVECs were grown to confluency in 24-well plates corning 6.5 mm Transwell inserts with 0.4 µm polycarbonate membranes in the upper chambers (inserts) (18312002) (Corning Incorporated, Corning, NY). Inserts were prepared with medium and flow-through was collected in order to check membranes. If no flow-through was found, inserts were put onto a new 24-well plate and HUVECs were treated with BIFO for 1 h. Then HUVECs were treated with 1 U/ml Thrombin for 24 h. Streptavidin-horseradish peroxidase (HRP) was added to the upper chamber and flow-through was collected from the lower chamber at indicated time points. Leakage of cell monolayers was quantified by the concentration of HRP in the lower chamber by photometric reading at 450 nm. Further details on measuring leakage with a Transwell-Assay are described elsewhere [39].
Statistical Analysis
We used GraphPad Prism5 (La Colla, CA) for data analysis and graph generation. Data was tested for Gaussian distribution with Kolmogorov-Smirnov-test. When data showed Gaussian distribution unpaired t-test with Welsh’s correction was used for comparison of two independent groups or One-Way-ANOVA with Bonferroni post test was used for comparison of more than two groups. When data did not show Gaussian distribution Mann-Whitney U test was used for comparison of two independent groups or One-Way-ANOVA with Dunn’s post test was used for statistical comparison of more than two independent groups. Results were seen as significant for p < 0.05. Columns are presented as mean ± SD.